Production and Purification of Esters of Polyunsaturated Fatty Acids

ABSTRACT

The present invention includes methods for producing and purifying esters of polyunsaturated fatty acids that include reacting a composition having triglycerides with polyunsaturated fatty acid residues in the presence of an alcohol and a base to produce an ester of a polyunsaturated fatty acid from the triglycerides. The composition can be a polyunsaturated fatty acid-containing composition that has not been conventionally processed. The reacted composition can be further processed by distillation.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority under 35 U.S.C. § 119(e)of U.S. Provisional Application No. 60/947,284, filed Jun. 29, 2007, thedisclosure of which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The invention relates to methods for producing and purifying esters ofpolyunsaturated fatty acids from triglyceride containing compositions.The invention also relates to compositions comprising polyunsaturatedfatty acids.

BACKGROUND OF THE INVENTION

It is desirable to increase the dietary intake of many beneficialnutrients. Particularly beneficial nutrients include fatty acids such asomega-3 and omega-6 long chain polyunsaturated fatty acids (LC-PUFAs)and esters thereof. Omega-3 PUFAs are recognized as important dietarycompounds for preventing arteriosclerosis and coronary heart disease,for alleviating inflammatory conditions and for retarding the growth oftumor cells. Omega-6 PUFAs serve not only as structural lipids in thehuman body, but also as precursors for a number of factors ininflammation, such as prostaglandins and leukotrienes. Long chainomega-3 and the omega-6 PUFAs represent important classes of PUFAs.

There are two main series or families of LC-PUFAs, depending on theposition of the double bond closest to the methyl end of the fatty acid:the omega-3 series contains a double bond at the third carbon, while theomega-6 series has no double bond until the sixth carbon. Thus,docosahexaenoic acid (“DHA”) has a chain length of 22 carbons with 6double bonds beginning with the third carbon from the methyl end and isdesignated “22:6 n-3”. Other important omega-3 LC-PUFAs includeeicosapentaenoic acid (“EPA”), which is designated “20:5 n-3,” andomega-3 docosapentaenoic acid (“DPA n-3”), which is designated “22:5n-3.” Important omega-6 LC-PUFAs include arachidonic acid (“ARA”), whichis designated “20:4 n-6,” and omega-6 docosapentaenoic acid (“DPA n-6”),which is designated “22:5 n-6.”

Because humans and many other animals cannot directly synthesize omega-3and omega-6 essential fatty acids, they must be obtained in the diet.Traditional dietary sources of PUFAs include vegetable oils, marineanimal oils, fish oils and oilseeds. In addition, oils produced bycertain microorganisms have been found to be rich in LC-PUFAs. The oilsderived from each of these sources, however, also contain substantiallevels of saturated fatty acids and other undesirable impurities.

Numerous methods have been used to isolate or purify PUFAs andderivatives thereof from crude oils. Among these processes arefractional crystallization at low temperatures, urea adductcrystallization, extraction with metal salt solutions, super criticalfluid fractionation on countercurrent columns and high performanceliquid chromatography.

The increased use of PUFAs and esters thereof in the fields of medicineand nutrition has created a commensurate need for PUFAs that areconcentrated and free of impurities. Previous efforts directed topurifying PUFAs, however, have suffered from problems such as high costsand decreased yields due, in part, to the use of harsh reagents.Accordingly, there is a need for improved methods of isolating andpurifying PUFAs in a form that can be consumed and utilized by humansand other animals.

SUMMARY OF THE INVENTION

The present invention provides a method for purifying a compositioncomprising triglycerides having polyunsaturated fatty acid residuescomprising reacting the composition in the presence of an alcohol and abase to produce an ester of a polyunsaturated fatty acid from thetriglycerides and distilling the composition to recover a fractioncomprising the ester of the polyunsaturated fatty acid.

In some embodiments, the step of reacting the composition in thepresence of an alcohol and a base is performed at a temperature fromabout 60° C. to about 120° C.

In some embodiments, the step of reacting the composition in thepresence of an alcohol and a base is performed for a time from about 2hours to about 12 hours.

In some embodiments, the composition comprising triglycerides havingpolyunsaturated fatty acid residues has not been subjected to one ormore treatments selected from the group consisting of refining,desolventization, deodorization, winterization, chill filtration, andbleaching.

In some embodiments, the composition comprising triglycerides havingpolyunsaturated fatty acid residues has not been subjected to refining,desolventization, deodorization, winterization, chill filtration, andbleaching.

In some embodiments, the composition comprising triglycerides havingpolyunsaturated fatty acid residues is from a source selected from thegroup consisting of a plant, a microorganism, an animal, and mixtures ofthe foregoing.

In some embodiments, the source is a microorganism selected from thegroup consisting of algae, bacteria, fungi and protists.

In some embodiments, the source is selected from the group consisting ofplants selected from the group consisting of soybean, corn, rice,safflower, sunflower, canola, flax, peanut, mustard, rapeseed, chickpea,cotton, lentil, white clover, olive, palm, borage, evening primrose,linseed and tobacco and mixtures thereof.

In some embodiments, the source is selected from the group consisting ofa genetically modified plant and a genetically modified microorganism,wherein the genetic modification comprises the introduction ofpolyketide synthase genes.

In some embodiments, the source is a microorganism selected from thegroup consisting of Thraustochytriales, dinoflagellates, andMortierella.

In some embodiments, the microorganism is Thraustochytriales,Schizochytrium or Thraustochytrium.

In some embodiments, the microorganism is a dinoflagellate of the genusCrypthecodinium.

In some embodiments, the source is an animal selected from aquaticanimals.

In some embodiments, the polyunsaturated fatty acid is a polyunsaturatedfatty acid having a chain length of at least 18 carbons.

In some embodiments, the polyunsaturated fatty acid is a polyunsaturatedfatty acid selected from the group consisting of docosahexaenoic acid,docosapentaenoic acid, arachidonic acid, eicosapentaenoic acid,stearidonic acid, linolenic acid, alpha linolenic acid, gamma linolenicacid, conjugated linolenic acid and mixtures thereof.

In some embodiments, the polyunsaturated fatty acid is docosahexaenoicacid.

In some embodiments, the polyunsaturated fatty acid is arachadonic acid.

In some embodiments, the base is a base of the formula RO-M, wherein Mis a monovalent cation and RO is an alkoxide of a C₁₋₆ alkyl alcohol.

In some embodiments, the base is sodium ethoxide.

In some embodiments, the alcohol is a C₁₋₆ alkyl alcohol.

In some embodiments, the alcohol is ethanol and the ester is an ethylester of the polyunsaturated fatty acid.

In some embodiments, the step of distilling the composition to recover afraction comprising the ester of the polyunsaturated fatty acid isperformed under vacuum.

In some embodiments, the step of distilling the composition to recover afraction comprising the ester of the polyunsaturated fatty acid isperformed at a temperature of less than about 170° C.

In some embodiments, the fraction recovered comprises at least about 50wt. %, 75 wt. %, 90 wt. %, or 95 wt. % ester of the polyunsaturatedfatty acid.

In some embodiments, the step of reacting the composition in thepresence of an alcohol and a base produces an ester of a polyunsaturatedfatty acid from the triglycerides by direct transesterification.

In some embodiments, the method further comprises a) combining thefraction comprising the ester of the polyunsaturated fatty acid withurea in a medium; b) cooling or concentrating the medium to form aurea-containing precipitate and a liquid fraction; and c) separating theprecipitate from the liquid fraction.

In some embodiments, the medium further comprises an organic solventthat can solubilize the ester of the polyunsaturated fatty acid.

In some embodiments, the organic solvent comprises an alkyl alcoholcomprising from 1 to 4 carbon atoms.

In some embodiments, the organic solvent comprises ethanol.

In some embodiments, the medium is cooled to a temperature of from about0° C. to about 25° C. to form the urea-containing precipitate.

In some embodiments, at least a portion of the urea-containingprecipitate is formed under a non-oxidizing atmosphere.

The present invention also provides a method for producing an ester of apolyunsaturated fatty acid from a composition comprising triglycerideshaving polyunsaturated fatty acid residues comprising transesterifyingthe composition in the presence of an alcohol and a base to produce anester of the polyunsaturated fatty acid from the triglycerides anddistilling the composition to recover a fraction comprising the ester ofthe polyunsaturated fatty acid.

The present invention further provides a method for purifying acomposition comprising triglycerides having polyunsaturated fatty acidresidues comprising reacting the composition in the presence of analcohol and a base to produce an ester of the polyunsaturated fatty acidfrom the triglycerides and separating a fraction comprising at leastabout 75% ester of the polyunsaturated fatty acid.

In some embodiments, the step of separating comprises distilling.

The present invention also provides a method for preparing a compositioncomprising an ester of a polyunsaturated fatty acid comprising reactinga composition comprising triglycerides having polyunsaturated fatty acidresidues in the presence of an alcohol and a base to produce an ester ofa polyunsaturated fatty acid from the triglycerides, wherein thecomposition comprising triglycerides having polyunsaturated fatty acidresidues has not been subjected to one or more treatments selected fromthe group consisting of refining, desolventization, deodorization,winterization, chill filtration, and bleaching.

In some embodiments, the step of reacting the composition in thepresence of an alcohol and a base is performed at a temperature fromabout 60° C. to about 120° C.

In some embodiments, the step of reacting the composition in thepresence of an alcohol and a base is performed for a time from about 2hours to about 12 hours.

In some embodiments, the method further comprises a step of distillingthe composition to recover a fraction comprising the ester of thepolyunsaturated fatty acid.

In some embodiments, the composition comprising triglycerides havingpolyunsaturated fatty acid residues is from a source selected from thegroup consisting of a plant, a microorganism, an animal, and mixtures ofthe foregoing.

In some embodiments, the source is a microorganism selected from thegroup consisting of algae, bacteria, fungi and protists.

In some embodiments, the source is selected from the group consisting ofplants selected from the group consisting of soybean, corn, rice,safflower, sunflower, canola, flax, peanut, mustard, rapeseed, chickpea,cotton, lentil, white clover, olive, palm, borage, evening primrose,linseed and tobacco and mixtures thereof.

In some embodiments, the source is selected from the group consisting ofa genetically modified plant and a genetically modified microorganism,wherein the genetic modification comprises the introduction ofpolyketide synthase genes.

In some embodiments, the source is a microorganism selected from thegroup consisting of Thraustochytriales, dinoflagellates, andMortierella.

In some embodiments, the source is a microorganism selected from thegroup consisting of Thraustochytriales, dinoflagellates, andMortierella.

In some embodiments, the microorganism is a dinoflagellate of the genusCrypthecodinium.

In some embodiments, the source is an animal selected from aquaticanimals.

In some embodiments, the polyunsaturated fatty acid is a polyunsaturatedfatty acid having a chain length of at least 18 carbons.

In some embodiments, the polyunsaturated fatty acid is a polyunsaturatedfatty acid selected from the group consisting of docosahexaenoic acid,docosapentaenoic acid, arachidonic acid, eicosapentaenoic acid,stearidonic acid, linolenic acid, alpha linolenic acid, gamma linolenicacid, conjugated linolenic acid and mixtures thereof.

In some embodiments, the polyunsaturated fatty acid is docosahexaenoicacid.

In some embodiments, the polyunsaturated fatty acid is arachadonic acid.

In some embodiments, the base is a base of the formula RO-M, wherein Mis a monovalent cation and RO is an alkoxide of a C₁₋₆ alkyl alcohol.

In some embodiments, the base is sodium ethoxide.

In some embodiments, the alcohol is a C₁₋₆ alkyl alcohol.

In some embodiments, the alcohol is ethanol and the ester is an ethylester of the polyunsaturated fatty acid.

In some embodiments, the step of distilling the composition to recover afraction comprising the ester of the polyunsaturated fatty acid isperformed under vacuum.

In some embodiments, the step of distilling the composition to recover afraction comprising the ester of the polyunsaturated fatty acid isperformed at a temperature of less than about 170° C.

In some embodiments, the fraction recovered comprises at least about 50wt. %, 75 wt. %, 90 wt. %, or 95 wt. % ester of the polyunsaturatedfatty acid.

In some embodiments, the step of reacting the composition in thepresence of an alcohol and a base produces an ester of a polyunsaturatedfatty acid from the triglycerides by direct transesterification.

The present invention also provides a composition comprising at leastabout 90 wt. % ethyl ester of docosahexaenoic acid, wherein thecomposition further comprises at least about 0.1 wt. % of4,7,10,13,16,19,22,25 octacosaoctaenoic acid (C28:8) or an esterthereof.

In some embodiments, the composition comprises at least about 0.5 wt. %,1.0 wt. %, or 1.2 wt. % of 4,7,10,13,16,19,22,25 octacosaoctaenoic acid(C28:8) or an ester thereof.

In some embodiments, the composition further comprises at least about0.1 wt. %, 0.3 wt. %, 0.4 wt. %, or 0.5 wt. % of docosapentaenoic acid(n-3) or an ester thereof.

In some embodiments, the composition comprises at least about 92 wt. %or 95 wt. % ethyl ester of docosahexaenoic acid.

In some embodiments, the composition further comprises less than about 1wt. %, 0.5 wt. % or 0.25 wt. % eicosapentaenoic acid or an esterthereof.

The present invention further provides a composition comprising at leastabout 90 wt. % ethyl ester of docosahexaenoic acid, wherein thecomposition further comprises at least about 0.1 wt. % ofdocosapentaenoic acid (n-3) or an ester thereof.

In some embodiments, the composition comprises at least about 0.3 wt. %,0.4 wt. %, or 0.5 wt. % of docosapentaenoic acid (n-3) or an esterthereof.

In some embodiments, the composition further comprises at least about0.5 wt. %, 0.75 wt. %, 1.0 wt. %, or 1.2 wt. % of 4,7,10,13,16,19,22,25octacosaoctaenoic acid (C28:8) or an ester thereof.

In some embodiments, the composition comprises at least about 92 wt. %or 95 wt. % ethyl ester of docosahexaenoic acid.

In some embodiments, the composition further comprises less than about 1wt. %, 0.5 wt. % or 0.25 wt. % eicosapentaenoic acid or an esterthereof.

The present invention also provides a composition comprising at leastabout 90 wt. % ethyl ester of docosahexaenoic acid, wherein thecomposition further comprises at least one additional fatty acid or anester thereof with a boiling point of about 150-170° C. at a pressure of0.8 mm Hg.

The present invention further provides a composition comprising at leastabout 70 wt. % ethyl ester of docosahexaenoic acid and at least about 25wt. % ethyl ester of docosapentaenoic acid (n-6).

In some embodiments, the composition further comprises less than about4% of a saturated fatty acid or an ester thereof.

In some embodiments, the saturated fatty acid or an ester thereofcontains less than 20 carbons.

In some embodiments, the saturated fatty acid or an ester thereofcontains 14 or 16 carbons.

The present invention also provides a composition comprising at leastabout 90 wt. % of a combination of ethyl ester of docosahexaenoic acidand ethyl ester of docosapentaenoic acid (n-6).

In some embodiments, the composition comprises at least about 10 wt. %ethyl ester of docosahexaenoic acid and at least about 10 wt. % ethylester of docosapentaenoic acid (n-6).

In some embodiments, the composition further comprises less than about4% of a saturated fatty acid or an ester thereof.

In some embodiments, the saturated fatty acid or an ester thereofcontains less than 20 carbons.

In some embodiments, the saturated fatty acid or an ester thereofcontains 14 or 16 carbons.

The present invention further provides a comprising at least about 90wt. % of a combination of ethyl ester of docosahexaenoic acid and ethylester of docosapentaenoic acid (n-6), wherein the composition furthercomprises at least one additional fatty acid or an ester thereof with aboiling point of about 150-175° C. at a pressure of 0.5 mm Hg.

The present invention also provides a method for preparing a compositioncomprising an ester of a polyunsaturated fatty acid comprising reactinga composition comprising triglycerides having polyunsaturated fatty acidresidues in the presence of an alcohol and a base to produce an ester ofa polyunsaturated fatty acid from the triglycerides, wherein thecomposition comprising triglycerides having polyunsaturated fatty acidresidues comprises at least one characteristic selected from the groupconsisting of: at least about 300 ppm phosphorus, at least about 0.4%free fatty acids, and a peroxide value of at least about 0.2 meq/kg.

The present invention also provides a method for purifying a compositioncomprising triglycerides having polyunsaturated fatty acid residues,wherein the composition comprises at least one characteristic selectedfrom the group consisting of: at least about 300 ppm phosphorus, atleast about 0.4% free fatty acids, and a peroxide value of at leastabout 0.2 meq/kg, comprising a) reacting the composition in the presenceof an alcohol and a base to produce an ester of a polyunsaturated fattyacid from the triglycerides; and b) distilling the composition torecover a fraction comprising the ester of the polyunsaturated fattyacid.

The present invention further provides a composition comprising at leastabout 60 wt. % esters of arachidonic acid.

In some embodiments, the composition further comprises less than about10 wt. % eicosapentaenoic acid.

In some embodiments, the esters of arachidonic acid are ethyl esters ofarachidonic acid.

The present invention also provides a method of treating a subject withhigh levels of triglycerides comprising administering a compositionaccording to claim 68, 81, 94, 95, 99 or 104 to the subject.

The present invention further provides a method of treating a subjectwith a neurological disorder, dementia or a pre-dementia relatedcondition comprising administering a composition according to claim 68,81, 94, 95, 99 or 104 to the subject.

DESCRIPTION OF THE INVENTION

The present invention provides novel methods for the purification ofcompositions containing triglycerides having PUFA residues. In variousaspects, the invention includes reacting the composition in the presenceof an alcohol and a base to produce an ester of a polyunsaturated fattyacid from the triglycerides. In one embodiment, the inventionadvantageously and efficiently is conducted on relatively crude oilsthat have not been subjected to conventional processing methods that caninclude refining, bleaching, deodorizing and winterization. In anotherembodiment, the invention includes producing esters from triglyceridesand then distilling the resulting composition to recover a fractioncomprising the ester of the polyunsaturated fatty acid. In an additionalembodiment, the fraction comprising the ester of the polyunsaturatedfatty acid is further purified by urea crystallization. The presentinvention allows the efficient and cost effective production of estersof PUFAs directly from crude or processed oils.

The starting material for the methods of the present invention is acomposition comprising triglycerides having PUFA residues. The terms“oils” and “compositions comprising triglycerides having PUFA residues”are used interchangeably throughout this application. As used herein, a“triglyceride” is an ester of three fatty acid residues and glycerolhaving a general chemical formula of CH₂(OOCR¹)CH(OOCR²)CH₂(OOCR³),wherein each of OOCR¹, OOCR², and OOCR³ represents a fatty acid residue.Suitable triglycerides contain at least one PUFA. In some embodiments,the PUFA has a chain length of at least 18 carbons. Such PUFAs arereferred to herein as long chain PUFAs or LC PUFAs. In some embodiments,the PUFA can be docosahexaenoic acid C22:6 n-3 (DHA), omega-3docosapentaenoic acid C22:5 n-3 (DPA), omega-6 docosapentaenoic acidC22:5 n-6 (DPA), arachidonic acid C20:4 n-6 (ARA), eicosapentaenoic acidC20:5 n-3 (EPA), stearidonic acid (SDA), linolenic acid (LLA), alphalinolenic acid (ALA), gamma linolenic acid (GLA), conjugated linolenicacid (CLA) or mixtures thereof. The PUFAs can also be present in any ofthe common forms found in natural lipids including but not limited totriacylglycerols, diacylglycerols, monoacylglycerols, phospholipids,free fatty acids, or in natural or synthetic derivative forms of thesefatty acids (e.g. calcium salts of fatty acids, and the like). Referenceto an oil or other composition comprising triglycerides having PUFAresidues, as used in the present invention, can refer to either acomposition comprising triglycerides having only a single type of LCPUFA residue such as DHA or a composition comprising triglycerideshaving a mixture of more than one type of LC PUFA residues such as morethan one of DHA, EPA and ARA.

Compositions comprising triglycerides having PUFA residues can beobtained from or derived from any suitable source, such as a plant(including oilseeds), a microorganism, an animal, or mixtures of theforegoing. The microorganisms can be algae, bacteria, fungi or protists.Microbial sources and methods for growing microorganisms comprisingnutrients and/or PUFAs are known in the art (Industrial Microbiology andBiotechnology, 2nd edition, 1999, American Society for Microbiology).For example, the microorganisms can be cultured in a fermentation mediumin a fermentor. Oils produced by microorganisms can be used in themethods and compositions of the present invention. In some embodiments,organisms include those selected from the group consisting of goldenalgae (such as microorganisms of the kingdom Stramenopiles), greenalgae, diatoms, dinoflagellates (such as microorganisms of the orderDinophyceae including members of the genus Crypthecodinium such as, forexample, Crypthecodinium cohnii), yeast, and fungi of the genera Mucorand Mortierella, including but not limited to Mortierella alpina andMortierella sect. schmuckeri. Members of the microbial groupStramenopiles include microalgae and algae-like microorganisms,including the following groups of microorganisms: Hamatores,Proteromonads, Opalines, Develpayella, Diplophrys, Labrinthulids,Thraustochytrids, Biosecids, Oomycetes, Hypochytridiomycetes, Commation,Reticulosphaera, Pelagomonas, Pelagococcus, Ollicola, Aureococcus,Parmales, Diatoms, Xanthophytes, Phaeophytes (brown algae),Eustigmatophytes, Raphidophytes, Synurids, Axodines (includingRhizochromulinaales, Pedinellales, Dictyochales), Chrysomeridales,Sarcinochrysidales, Hydrurales, Hibberdiales, and Chromulinales. TheThraustochytrids include the genera Schizochytrium (species includeaggregatum, limnaceum, mangrovei, minutum, octosporum), Thraustochytrium(species include arudimentale, aureum, benthicola, globosum, kinnei,motivum, multirudimentale, pachydermum, proliferum, roseum, striatum),Ulkenia (species include amoeboidea, kerguelensis, minuta, profunda,radiate, sailens, sarkariana, schizochytrops, visurgensis, yorkensis),Aplanochytrium (species include haliotidis, kerguelensis, profunda,stocchinoi), Japonochytrium (species include marinum), Althornia(species include crouchii), and Elina (species include marisalba,sinorifica). The Labrinthulids include the genera Labyrinthula (speciesinclude algeriensis, coenocystis, chattonii, macrocystis, macrocystisatlantica, macrocystis macrocystis, marina, minuta, roscoffensis,valkanovii, vitellina, vitellina pacifica, vitellina vitellina, zopfi),Labyrinthomyxa (species include marina), Labyrinthuloides (speciesinclude haliotidis, yorkensis), Diplophrys (species include archeri),Pyrrhosorus* (species include marinus), Sorodiplophrys* (species includestercorea), Chlamydomyxa* (species include labyrinthuloides, montana).(*=there is no current general consensus on the exact taxonomicplacement of these genera).

Suitable microorganisms include those capable of producing lipidscomprising omega-3 and/or omega-6 polyunsaturated fatty acids, and inparticular microorganisms that are capable of producing oils containingDHA, DPA, EPA or ARA will be described. More particularly, preferredmicroorganisms are algae, such as Thraustochytrids of the orderThraustochytriales, including Thraustochytrium (including Ulkenia) andSchizochytrium and including Thraustochytriales which are disclosed incommonly assigned U.S. Pat. Nos. 5,340,594 and 5,340,742, both issued toBarclay, all of which are incorporated herein by reference in theirentirety. More preferably, the microorganisms are selected from thegroup consisting of microorganisms having the identifyingcharacteristics of ATCC number 20888, ATCC number 20889, ATCC number20890, ATCC number 20891 and ATCC number 20892. Since there is somedisagreement among experts as to whether Ulkenia is a separate genusfrom the genus Thraustochytrium, for the purposes of this application,the genus Thraustochytrium will include Ulkenia. Also preferred arestrains of Mortierella schmuckeri (e.g., including ATCC 74371) andMortierella alpina. Also preferred are strains of Crypthecodiniumcohnii, including microorganisms having the identifying characteristicsof ATCC Nos. 30021, 30334-30348, 30541-30543, 30555-30557, 30571, 30572,30772-30775, 30812, 40750, 50050-50060, and 50297-50300. Oleaginousmicroorganisms are also preferred. As used herein, “oleaginousmicroorganisms” are defined as microorganisms capable of accumulatinggreater than 20% of the dry weight of their cells in the form of lipids.Genetically modified microorganisms that produce PUFA-containing oilsare also suitable for the present invention. These can include naturallyPUFA-producing microorganisms that have been genetically modified aswell as microorganisms that do not naturally produce PUFAs but that havebeen genetically modified to do so.

Suitable organisms can be obtained from a number of available sources,including by collection from the natural environment. For example, theAmerican Type Culture Collection currently lists many publicly availablestrains of microorganisms identified above. As used herein, anyorganism, or any specific type of organism, includes wild strains,mutants, or recombinant types. Growth conditions in which to culture orgrow these organisms are known in the art, and appropriate growthconditions for at least some of these organisms are disclosed in, forexample, U.S. Pat. No. 5,130,242, U.S. Pat. No. 5,407,957, U.S. Pat. No.5,397,591, U.S. Pat. No. 5,492,938, U.S. Pat. No. 5,711,983 and U.S.Pat. No. 6,607,900, all of which are incorporated herein by reference intheir entirety. When microbial oils are used, the microorganisms arecultured in an effective medium, herein defined as any medium capable ofpromoting oil production. Preferably, the effective medium also promotesrapid microbial growth. The microorganisms can be cultured inconventional fermentation modes, which include, but are not limited to,batch, fed-batch, and continuous.

Another source of oils suitable for the compositions and methods of thepresent invention includes a plant source, such as oilseed plants.PUFA-producing plants, in alternate embodiments, can include thosegenetically engineered to express genes that produce PUFAs and thosethat produce PUFAs naturally. Such genes can include genes encodingproteins involved in the classical fatty acid synthase pathways, orgenes encoding proteins involved in the PUFA polyketide synthase (PKS)pathway. The genes and proteins involved in the classical fatty acidsynthase pathways, and genetically modified organisms, such as plants,transformed with such genes, are described, for example, in Napier andSayanova, Proceedings of the Nutrition Society (2005), 64:387-393;Robert et al., Functional Plant Biology (2005) 32:473-479; or U.S.Patent Application Publication 2004/0172682. The PUFA PKS pathway, genesand proteins included in this pathway, and genetically modifiedmicroorganisms and plants transformed with such genes for the expressionand production of PUFAs are described in detail in: U.S. Pat. No.6,140,486, U.S. Pat. No. 6,566,583; U.S. Patent Application PublicationNo. 20020194641, U.S. Pat. No. 7,211,418, U.S. Patent ApplicationPublication No. 20050100995A1, U.S. Patent Application Publication No.20070089199, PCT Publication No. WO 05/097982, and U.S. PatentApplication Publication No. 20050014231, each of which is incorporatedherein by reference in its entirety.

Oilseed crops suitable for use in the present invention includesoybeans, corn, rice, safflower, sunflower, canola, flax, peanut,mustard, rapeseed, chickpea, cotton, lentil, white clover, olive, palmoil, borage, evening primrose, linseed, and tobacco that have beengenetically modified to produce a PUFA as described above.

Genetic transformation techniques for microorganisms and plants arewell-known in the art. Transformation techniques for microorganisms arewell known in the art and are discussed, for example, in Sambrook etal., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLabs Press. A general technique for transformation of dinoflagellates,which can be adapted for use with Crypthecodinium cohnii, is describedin detail in Lohuis and Miller, The Plant Journal (1998) 13(3): 427-435.A general technique for genetic transformation of Thraustochytrids isdescribed in detail in U.S. Patent Application Publication No.20030166207, published Sep. 4, 2003. Methods for the genetic engineeringof plants are also well known in the art. For instance, numerous methodsfor plant transformation have been developed, including biological andphysical transformation protocols. See, for example, Miki et al.,“Procedures for Introducing Foreign DNA into Plants” in Methods in PlantMolecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E.Eds. (CRC Press, Inc., Boca Raton, 1993) pp. 67-88. In addition, vectorsand in vitro culture methods for plant cell or tissue transformation andregeneration of plants are available. See, for example, Gruber et al.,“Vectors for Plant Transformation” in Methods in Plant Molecular Biologyand Biotechnology, Glick, B. R. and Thompson, J. E. Eds. (CRC Press,Inc., Boca Raton, 1993) pp. 89-119. See also, Horsch et al., Science227:1229 (1985); Kado, C. I., Crit. Rev. Plant. Sci. 10:1 (1991);Moloney et al., Plant Cell Reports 8:238 (1989); U.S. Pat. No.4,940,838; U.S. Pat. No. 5,464,763; Sanford et al., Part. Sci. Technol.5:27 (1987); Sanford, J. C., Trends Biotech. 6:299 (1988); Sanford, J.C., Physiol. Plant 79:206 (1990); Klein et al., Biotechnology 10:268(1992); Zhang et al., Bio/Technology 9:996 (1991); Deshayes et al., EMBOJ., 4:2731 (1985); Christou et al., Proc Natl. Acad. Sci. USA 84:3962(1987); Hain et al., Mol. Gen. Genet. 199:161 (1985); Draper et al.,Plant Cell Physiol. 23:451 (1982); Donn et al., In Abstracts of VIIthInternational Congress on Plant Cell and Tissue Culture IAPTC, A2-38, p.53 (1990); D'Halluin et al., Plant Cell 4:1495-1505 (1992) and Spenceret al., Plant Mol. Biol. 24:51-61 (1994).

When oilseed plants are the source of PUFA-containing oils, the seedscan be harvested and processed to remove any impurities, debris orindigestible portions from the harvested seeds. Processing steps varydepending on the type of oilseed and are known in the art. Processingsteps can include threshing (such as, for example, when soybean seedsare separated from the pods), dehulling (removing the dry outercovering, or husk, of a fruit, seed, or nut), drying, cleaning,grinding, milling and flaking. After the seeds have been processed toremove any impurities, debris or indigestible materials, they can beadded to an aqueous solution and then mixed to produce a slurry. In someembodiments, milling, crushing or flaking is performed prior to mixingwith water. A slurry produced in this manner can be treated andprocessed the same way as described for a microbial fermentation broth.

Another biomass source of PUFA-containing oils suitable for thecompositions and methods of the present invention includes an animalsource. Examples of animal sources include aquatic animals (e.g., fish,marine mammals, and crustaceans such as krill and other euphausids) andanimal tissues (e.g., brain, liver, eyes, etc.) and animal products suchas eggs or milk. Techniques for recovery of PUFA-containing oils fromsuch sources are known in the art.

While in one embodiment of the invention the composition comprisingtriglycerides having PUFA residues can be a crude oil (discussed in moredetail below), other such compositions useful in the present inventioncan be recovered from their sources by any suitable means known to thosein the art. For example, oils can be recovered by extraction withsolvents such as chloroform, hexane, methylene chloride, methanol andthe like, or by supercritical fluid extraction. Alternatively, the oilscan be extracted using extraction techniques, such as are described inU.S. Pat. No. 6,750,048 and PCT Patent Application Serial No.US01/01806, both filed Jan. 19, 2001, and entitled “SolventlessExtraction Process,” both of which are incorporated herein by referencein their entirety. Additional extraction and/or purification techniquesare taught in PCT Patent Application Serial No. PCT/IB01/00841 entitled“Method for the Fractionation of Oil and Polar Lipid-Containing NativeRaw Materials” filed Apr. 12, 2001; PCT Patent Application Serial No.PCT/IB01/00963 entitled “Method for the Fractionation of Oil and PolarLipid-Containing Native Raw Materials Using Water-Soluble OrganicSolvent and Centrifugation” filed Apr. 12, 2001; U.S. Provisional PatentApplication Ser. No. 60/291,484 entitled “Production and Use of a PolarLipid-Rich Fraction Containing Stearidonic Acid and Gamma Linolenic Acidfrom Plant Seeds and Microbes filed May 14, 2001; U.S. ProvisionalPatent Application Ser. No. 60/290,899 entitled “Production and Use of aPolar-Lipid Fraction Containing Omega-3 and/or Omega-6 HighlyUnsaturated Fatty Acids from Microbes, Genetically Modified Plant Seedsand Marine Organisms” filed May 14, 2001; U.S. Pat. No. 6,399,803entitled “Process for Separating a Triglyceride Comprising aDocosahexaenoic Acid Residue from a Mixture of Triglycerides” issuedJun. 4, 2002 filed Feb. 17, 2000; and PCT Patent Application Serial No.US01/01010 entitled “Process for Making an Enriched Mixture ofPolyunsaturated Fatty Acid Esters” filed Jan. 11, 2001; all of which areincorporated herein by reference in their entirety. The extracted oilscan be evaporated under reduced pressure to produce a sample ofconcentrated oil material. Processes for the enzyme treatment of biomassfor the recovery of lipids are disclosed in U.S. Provisional PatentApplication No. 60/377,550, entitled “HIGH-QUALITY LIPIDS AND METHODSFOR PRODUCING BY ENZYMATIC LIBERATION FROM BIOMASS,” filed on May 3,2002; PCT Patent Application Serial No. PCT/US03/14177 entitled“HIGH-QUALITY LIPIDS AND METHODS FOR PRODUCING BY ENZYMATIC LIBERATIONFROM BIOMASS,” filed on May 5, 2003; copending U.S. patent applicationSer. No. 10/971,723, entitled “HIGH-QUALITY LIPIDS AND METHODS FORPRODUCING BY LIBERATION FROM BIOMASS,” filed on Oct. 22, 2004; EP PatentPublication 0 776 356 and U.S. Pat. No. 5,928,696, both entitled“Process for extracting native products which are not water-soluble fromnative substance mixtures by centrifugal force,” the disclosures ofwhich are hereby incorporated by reference herein in their entirety.

In some embodiments, an oil obtained from a source described above canserve as the starting material for the methods of the present inventioneven when it has not been subjected to conventional processing. Examplesof such conventional processes that may be avoided include refining(e.g., physical refining, silica refining or caustic refining),desolventization, deodorization, winterization, chill filtration, and/orbleaching. Thus, in certain embodiments, the composition containingtriglycerides having PUFA residues has not been subjected to one or moretreatments selected from refining, desolventization, deodorization,winterization, chill filtration, and bleaching and in furtherembodiments, the composition has not been subjected to any one ofrefining, desolventization, deodorization, winterization, chillfiltration, and bleaching.

In further aspects of the invention, the composition comprisingtriglycerides having polyunsaturated fatty acid residues may be an oilhaving characteristics of oils that have not been subjected toconventional processing, such as refining, desolventization,deodorization, winterization, chill filtration, and bleaching. Thus, asuitable oil can have a chemical or physical characteristic of anunprocessed oil. For example, the oil may contain an undesirablecomponent (e.g., an impurity) at a level that is typically not presentin a conventionally processed oil. For example, the oil may contain fromabout 300 ppm phosphorous to about 1000 ppm phosphorous. In someembodiments, the oil comprises at least about 300 ppm phosphorous; atleast about 400 ppm phosphorous; at least about 500 ppm phosphorous; atleast about 600 ppm phosphorous; at least about 650 ppm phosphorous; atleast about 700 ppm phosphorous; at least about 750 ppm phosphorous; atleast about 800 ppm phosphorous; at least about 850 ppm phosphorous; atleast about 900 ppm phosphorous; at least about 950 ppm phosphorous; orat least about 1000 ppm phosphorous. In another aspect, the oil maycontain free fatty acids in a range of from about 0.4 wt. % to about 1.4wt. %. In certain embodiments, the oil comprises at least about 0.4 wt.% free fatty acids; at least about 0.6 wt. % free fatty acids; at leastabout 0.8 wt. % free fatty acids; at least about 0.9 wt. % free fattyacids; at least about 1.0 wt. % free fatty acids; at least about 1.1 wt.% free fatty acids; at least about 1.2 wt. % free fatty acids; at leastabout 1.3 wt. % free fatty acids; or at least about 1.4 wt. % free fattyacids. In another aspect, the oil may contain a peroxide value rangingfrom about 0.2 meq/kg to about 2.5 meq/kg. In some embodiments, the oilcomprises a peroxide value of at least about 0.2 meq/kg; a peroxidevalue of at least about 0.4 meq/kg; a peroxide value of at least about0.6 meq/kg; a peroxide value of at least about 0.8 meq/kg; a peroxidevalue of at least about 1.0 meq/kg; a peroxide value of at least about1.2 meq/kg; a peroxide value of at least about 1.4 meq/kg; a peroxidevalue of at least about 1.5 meq/kg; a peroxide value of at least about1.6 meq/kg; a peroxide value of at least about 1.7 meq/kg; a peroxidevalue of at least about 1.8 meq/kg; a peroxide value of at least about1.9 meq/kg; a peroxide value of at least about 2.0 meq/kg; a peroxidevalue of at least about 2.1 meq/kg; a peroxide value of at least about2.2 meq/kg; a peroxide value of at least about 2.3 meq/kg; a peroxidevalue of at least about 2.4 meq/kg; or a peroxide value of at leastabout 2.5 meq/kg.

In some embodiments, the crude oil may be isolated from a microorganismusing standard techniques, without being subjected to further refinementor purification. In such embodiments, the oil is a microbial oil thathas only been subjected to solvent extraction, such as hexaneextraction, isopropanol extraction, or the like.

In other embodiments, compositions comprising triglycerides havingpolyunsaturated fatty acid residues, such as oils described above, maybe subjected to further processing steps, such as refining,desolventization, deodorization, winterization, chill filtration, and/orbleaching. Such “processed” oils include microbial oils that have beensubjected to solvent extraction and one or more of these additionalprocessing steps. In some embodiments, oils are minimally processed.“Minimally processed” oils include microbial oils that have beensubjected to solvent extraction and filtration. In certain embodiments,minimally processed oils are further subjected to winterization.

Methods of the present invention involve reacting compositionscontaining triglycerides having PUFA residues in the presence of analcohol and a base to produce esters of the PUFAs from thetriglycerides.

Alcohols suitable for use in the present invention include any loweralkyl alcohol containing from 1 to 6 carbon atoms (i.e., a C₁₋₆ alkylalcohol). Without being bound by theory, it is believed that the use oflower alkyl alcohols in the methods of the present invention produceslower alkyl esters of the PUFAs. For example, the use of ethanolproduces ethyl esters. In certain embodiments, the alcohol is methanolor ethanol. In these embodiments, the PUFA esters produced are a methylester and an ethyl ester of the PUFA, respectively. In processes of thepresent invention, the alcohol typically comprises between about 25 wt.% and about 50 wt. %; between about 30 wt. % and about 45 wt. %, orbetween about 35 wt. % and about 40 wt. % of the mixture of thecomposition, the alcohol and the base. In some embodiments, the alcoholcomprises about 38 wt. % of the mixture of the composition, the alcoholand the base. In certain embodiments, the composition and the base canbe added to either pure ethanol or pure methanol. In general, the amountof alcohol used may vary with the solubility of the oil or compositioncontaining triglycerides having PUFA residues in the alcohol.

Any base known in the art to be suitable for use as a reactant may beused in the present invention. Bases of the formula RO-M, wherein M is amonovalent cation and RO is an alkoxide of a C₁₋₆ alkyl alcohol areparticularly suited for the present invention. Examples of suitablebases include elemental sodium, sodium methoxide, sodium ethoxide,potassium methoxide, and potassium ethoxide. In some embodiments, thebase is sodium ethoxide. In processes of the present invention, the baseis typically added in an amount of between about 0.5 and about 1.5 molarequivalents of triglycerides, between about 0.7 and about 1.4 molarequivalents of triglycerides, between about 0.9 and about 1.3 molarequivalents of triglycerides, or between about 1.0 and about 1.2 molarequivalents of triglycerides to the reaction step with the compositionand the alcohol. In certain embodiments, the base is typically added inan amount of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.95,1.0, 1.01, 1.02, 1.03, 1.04, 1.05, 1.10, 1.15, 1.2, 1.3, 1.4, or 1.5molar equivalents of triglycerides to the reaction step with thecomposition and the alcohol. In some embodiments, the base is added inan amount of 1.04 molar equivalents of triglycerides to the reactionstep with the composition and the alcohol.

The composition comprising triglycerides having polyunsaturated fattyacid residues, the alcohol and the base are reacted together at atemperature and for an amount of time that allows the production of anester between the fatty acid residues and the alcohol. Suitable reactiontimes and temperatures may be determined by one of skill in the art toproduce an ester. Without intending to be bound by theory, the PUFAresidues are believed to be cleaved from the glycerol backbone of thetriglyceride and esters of each PUFA residue are formed during the stepof reacting. In certain embodiments, the step of reacting thecomposition in the presence of an alcohol and a base is performed at atemperature from about 60° C. to about 120° C., from about 70° C. toabout 110° C., from about 75° C. to about 100° C., or from about 80° C.to about 90° C. In further embodiments, the step of reacting thecomposition in the presence of an alcohol and a base is performed at atemperature of about 75° C., 80° C., 85° C., 90° C., or 95° C. In someembodiments, the step of reacting the composition in the presence of analcohol and a base is performed for a time from about 2 hours to about12 hours, from about 3 hours to about 11 hours, from about 4 hours toabout 10 hours, from about 5 hours to about 9 hours, or from about 6hours to about 8 hours. In certain embodiments, the step of reacting thecomposition in the presence of an alcohol and a base is performed forabout 5.5, 6, 6.5, 7, 7.5, 8, or 8.5 hours

In one embodiment, the step of reacting the oil composition, alcohol andbase may be conducted by refluxing the components to produce the PUFAesters. In additional embodiments, the step of reacting the oilcomposition may be carried out at a temperature that does not result inthe refluxing of the reaction components. For example, carrying out thestep of reacting the oil composition under pressures greater thanatmospheric pressure can increase the boiling point of the solventspresent in the reaction mixture. Under such conditions, the reaction canoccur at a temperature at which the solvents would boil at atmosphericpressure, but would not result in the refluxing of the reactioncomponents. In some embodiments, the reaction is conducted at a pressurefrom about 5 to about 20 pounds per square inch (psi); from about 7 toabout 15 psi; or from about 9 to about 12 psi. In certain embodiments,the reaction is conducted at a pressure of about 7, 8, 9, 10, 11, or 12psi. Reactions conducted under pressure may be carried out at thereaction temperatures listed above. In some embodiments, reactionsconducted under pressure may be carried out at about 70° C., 75° C., 80°C., 85° C., or 90° C.

The reaction mixture comprising PUFA esters can be further processed toobtain the PUFA esters from the mixture. For example, the mixture may becooled, diluted with water, and the aqueous solution extracted with asolvent such as hexane to produce a composition comprising PUFA esters.Techniques for washing and/or extracting crude reaction mixtures areknown in the art.

In one embodiment of the present invention, PUFA esters are separatedfrom the reaction mixture by distilling the composition to recover afraction comprising the ester of the polyunsaturated fatty acid. In thismanner, a targeted fraction of the reaction mixture including PUFAesters of interest can be separated from the reaction mixture andrecovered.

In certain embodiments, the distillation is performed under vacuum.Without being bound by theory, distillation under vacuum allows thedistillation to be accomplished at a lower temperature than in theabsence of a vacuum and thus may prevent the degradation of the esters.Typical distillation temperatures range from about 120° C. to about 170°C. In some embodiments, the step of distilling is performed at atemperature of less than about 180° C., less than about 175° C., lessthan about 170° C., less than about 165° C., less than about 160° C.,less than about 155° C., less than about 150° C., less than about 145°C., less than about 140° C., less than about 135° C., or less than about130° C. Typical pressures for vacuum distillation range from about 0.1mm Hg to about 10 mm Hg. In some embodiments, the pressure for vacuumdistillation is about 0.1, 0.5, 1, 1.5, 2, 2, 5, 3, 3.5, or 4 mm Hg.

The methods of the present invention may be used to produce compositionsthat contain a high percentage of PUFA esters. For example, suchcompositions can contain between about 50 wt. % and about 100 wt. % ofan ester of a PUFA, and in other embodiments, the composition cancomprise at least about 50 wt. %, at least about 55 wt. %, at leastabout 60 wt. %, at least about 65 wt. %, at least about 70 wt. %, atleast about 75 wt. %, at least about 80 wt. %, at least about 85 wt. %,at least about 90 wt. %, at least about 95 wt. %, at least about 99 wt.% of esters of a PUFA.

In some embodiments, the PUFA esters are subjected to a ureacrystallization step. When urea crystallizes in a solution containingPUFA esters (e.g., esters of DHA) and saturated fatty acid esters formedby the transesterification of a glyceride source using the techniquesdiscussed above, a precipitate forms that comprises the urea and atleast a portion of the saturated fatty acid esters. This precipitate,however, comprises a substantially lesser fraction of the PUFA estersthan the initial reaction mixture. The bulk of the PUFA esters insteadremain in solution and can therefore be easily separated from theprecipitated saturated fatty acid esters.

The urea crystallization separation process comprises first forming asolution comprising fatty acid esters and urea. The amount of ureapreferably is proportional to the total amount of saturated fatty acidsto be separated from the solution. When separating fatty acid estersfrom the transesterification reaction mixtures described above, the massratio of the mixture of fatty acid esters to urea is typically about1:2. The solution also preferably comprises an organic solvent that cansolubilize urea and the desired PUFA ester, and more preferably cansolubilize urea and all the fatty acid esters in the mixture. Examplesof suitable solvents include alkyl alcohols having from 1 to 4 carbons,with methanol and ethanol being more preferred, and ethanol being themost preferred. The volumetric ratio of the mixture of fatty acid estersto solvent is preferably about 1:10.

Essentially all the urea preferably is dissolved in the solution. Thismay generally be achieved by heating the solution. The solution,however, preferably is not heated to a temperature above the boilingpoint of the organic solvent. Typically, the solution is heated to atemperature of about 60° C., 65° C., 70° C., 75° C. or 80° C.

Once the urea is dissolved, the PUFA esters are added to the solution.Upon addition, if solids remain, the mixture may be heated until solidsdissolve. The solution may be cooled to form a precipitate comprisingurea adducts of fatty acid esters. In certain embodiments, the solutionis cooled to a temperature that is from about 0° C. to about 25° C.,such as from about 15° C. to about 25° C. In other embodiments, thesolution is cooled to a temperature of about 0° C. about 5° C. about 10°C. about 15° C., about 20° C., about 25° C., or from about 20° C. toabout 25° C. Once the solution is cooled, it may be allowed to stand fora period of time (typically no greater than about 20 hours) at thecooling temperature with occasional stirring.

In another embodiment of this invention, after the solution (comprisingfatty acid esters and dissolved urea) is formed, a precipitatecomprising urea is formed by concentrating the solution. The solutionmay be concentrated, for example, by evaporating a portion of thesolvent in the solution. The amount of solvent removed preferably issufficient to cause the urea concentration in the solution to exceed thesaturation concentration.

During the urea crystallization separation process, the solution may bekept in a non-oxidizing atmosphere, such as an atmosphere consistingessentially of a noble gas, N₂, or a combination thereof, with anatmosphere consisting essentially of N₂ being most preferred. Use ofsuch an atmosphere may aid in minimizing oxidation of carbon-carbondouble bonds of the PUFA esters.

After the precipitate comprising urea has formed, the precipitate may beseparated from the liquid fraction enriched in PUFA esters. This may beachieved, for example, by filtration or centrifugation. In oneembodiment, the precipitate may be subsequently washed with a smallquantity of the organic solvent (preferably saturated with urea) torecover any residual unprecipitated desired PUFA ester that remains withthe precipitate. This solvent, in turn, may be combined with the liquidfraction.

The liquid fraction may be concentrated, combined with water, and thenthe esters therein may be extracted with a non-polar solvent from theresulting mixture. The liquid fraction may be concentrated, for example,by evaporating a portion of the solvent from the liquid fraction (theamount of solvent evaporated, however, preferably is not so great as tocause further urea to precipitate). The amount of water subsequentlycombined with the resulting concentrated liquid fraction may varywidely. Preferably, the volume ratio of water to concentrated liquidfraction is about 2:1 (in a particularly preferred embodiment,sufficient acid (preferably H₂SO₄) is also introduced to neutralize theurea). The non-polar solvent that may be used to extract the fatty acidesters from the resulting concentrated-mother-liquor/water mixture maybe, for example, petroleum ether, pentane, hexane, cyclohexane, ethylacetate, or heptane, with hexane being the most preferred. Thevolumetric ratio of the non-polar solvent to theconcentrated-mother-liquor/water mixture preferably is about 2:3.

In other embodiments, the liquid fraction may also be extracted with aslightly polar organic solvent to maximize recovery of the fatty acidesters (which are slightly polar). Examples of suitable slightly polarsolvents include diethyl ether and ethyl acetate, with diethyl etherbeing most preferred. Preferably, the volumetric ratio of slightly polarsolvent to the mother-liquor/water mixture is about 2:3. Following theextraction with this slightly polar solvent, the solvent preferably maybe combined with the non-polar solvent used in the initial extraction.

After the extraction is complete, any residual water may be removed fromthe extraction solvent by, for example, washing the solvent with brineand/or passing the solvent over an anhydrous salt (e.g., sodiumsulfate). The solution then preferably is concentrated by, for example,evaporating a portion of the solvent.

By way of example, the methods of the present invention may be used topurify ethyl arachidonate (arachidonic acid ethyl ester) from a crudeMortierella alpina oil. A crude oil obtained from Mortierella alpina byhexane extraction (typically with an ARA content of about 0.5 g/g oil)can be used directly without any further processing, such aswinterization and/or RBD processing. 150 mL of absolute ethanol can beadded to 175 g (approximately 0.2 moles) of the crude oil in a one-literflask under N₂ at room temperature. The mixture can be allowed to stirfor 15 minutes to obtain a homogeneous solution. 67 g of a 21% solutionof NaOEt/EtOH (approximately 1.04 molar equivalents) can be then addedto the solution, and the mixture can be allowed to reflux under N₂ forabout 10 hours. The progress of the reaction may be monitored by gaschromatography (GC) and/or thin-layer chromatography (TLC).

When the reaction is completed, approximately 75 mL of ethanol can beremoved by distillation, and the mixture can be allowed to cool to roomtemperature under N₂. 300 mL of hexane can be added to the cooledmixture, and the mixture can be allowed to stir for 15 minutes at roomtemperature. 300 mL of deionized water can be then added to the mixture,and the mixture can be allowed to stir for an additional 15 minutes.After removing and saving the organic layer, the aqueous layer can bewashed twice with 300 mL portions of hexane. The combined organic layercan be washed with 200 mL of a saturated NaCl solution. A GC analysis ofthe organic layer may be used to determine the amount of ARA ethyl esterpresent in the crude product. In some embodiments, approximately 50% ofthe crude product is ARA ethyl ester, with the remaining materials beingpredominantly lower molecular weight ethyl esters. The crude product maythen be subjected to vacuum fractional distillation or otherpurification procedures. In certain embodiments, a purity of greaterthan about 60% ARA ethyl ester may be achieved following the fractionaldistillation of the crude product.

Without being bound by theory, it is believed that the methods of thepresent invention result in the direct transesterification oftriglycerides having PUFA residues to produce esters of the PUFAs.Previous methods utilized long reaction times, large amounts ofreagents, and subjected the oils to harsh conditions such as hightemperatures and highly acidic conditions. The methods disclosed hereinthus provide a more efficient and economical purification process thatyields a pure product. Furthermore, the methods disclosed herein may beapplied to crude oils as well as purified oils, resulting in anadditional increase in efficiency and cost savings.

Other embodiments of the present invention include compositions producedby the methods described herein. As noted above, such compositions cancontain greater than about 50 wt. %, greater than about 55 wt. %, etc.of esters of a PUFA. In such embodiments, the compositions can containat least about 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 wt. % ofPUFA esters. In other embodiments, the compositions may further compriseless than about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.25 or 0.1 wt. %eicosapentaenoic acid. The compositions of the invention may include anyPUFA esters as described above, namely, DHA, omega-3 DPA, omega-6 DPA,ARA, SDA, LLA, ALA, GLA, or CLA or combinations thereof. In someembodiments, the compositions may comprise ethyl esters. In certainembodiments, the composition comprises at least about 89 wt. % DHAesters. In other embodiments, the composition comprises at least about89 wt. % of a combination of DHA and DPA esters.

Compositions of the present invention also include compositions thatcontain at least about 60, 65, 70, 75, 80, 85, 90, or 95 wt. % ARAesters. In some embodiments, the ARA esters may be ethyl esters of ARA.In other embodiments, the compositions may further comprise less thanabout 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.25 or 0.1 wt. %eicosapentaenoic acid.

The present invention also provides compositions comprising at leastabout 90 wt. % ethyl ester of docosahexaenoic acid (DHA) and at leastabout 0.1 wt. % of 4,7,10,13,16,19,22,25 octacosaoctaenoic acid (C28:8).These compositions may be produced by the methods disclosed herein. Insome embodiments, the amount of ethyl ester of DHA in the compositionsmay be at least about 91, 92, 93, 94, 95, 96, 97, 98, or 99 wt. %. Incertain embodiments, the amount of C28:8 in the compositions may be atleast about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3,1.4 or 1.5 wt. %. The C28:8 may be present in triglyceride or esterform. For example, the C28:8 may be present in ethyl ester form.

The present invention also provides compositions comprising at leastabout 90 wt. % ethyl ester of docosahexaenoic acid (DHA) and at leastabout 0.1 wt % of DPA (n-3). These compositions may be produced by themethods disclosed herein. In some embodiments, the amount of ethyl esterof DHA in the compositions may be at least about 91, 92, 93, 94, 95, 96,97, 98, or 99 wt. %. In certain embodiments, the amount of DPA (n-3) inthe compositions may be at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, or 1.0 wt. % of DPA (n-3). The DPA (n-3) may be presentin triglyceride or ester form. For example, the DPA (n-3) may be presentin ethyl ester form.

In certain embodiments, the compositions comprise all three of the ethylester of DHA, C28:8 and DPA (n-3) in the concentration ranges specifiedabove.

In further embodiments, the compositions may comprise less than about1.0, 0.9, 0.8. 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 wt. % EPA inaddition to the ethyl ester of DHA and C28:8. In one embodiment, thecompositions may comprise less than about 0.25 wt. % EPA. The EPA may bepresent in triglyceride or ester form. For example, the EPA may bepresent in ethyl ester form. In some embodiments, the compositions maycomprise 0 wt. % EPA.

The present invention also provides compositions comprising at leastabout 90 wt. % ethyl ester of docosahexaenoic acid and at least oneadditional fatty acid or an ester thereof. In some embodiments, theamount of ethyl ester of DHA in the compositions may be at least about91, 92, 93, 94, 95, 96, 97, 98, or 99 wt. %. In certain embodiments, theadditional fatty acid may have a boiling point of about 150-170° C. at apressure of 0.8 mm Hg.

The present invention further includes compositions comprising at leastabout 70 wt. % ethyl ester of docosahexaenoic acid (DHA) and at leastabout 25 wt. % ethyl ester of docosapentaenoic acid (n-6).

Compositions of the present invention also include compositions thatcomprises at least about 90 wt. % of a combination of ethyl ester ofdocosahexaenoic acid and ethyl ester of docosapentaenoic acid (n-6). Incertain embodiments, the compositions may comprise at least about 91,92, 93, 94, 95, 96, 97, 98, or 99 wt. % of a combination of ethyl esterof docosahexaenoic acid and ethyl ester of docosapentaenoic acid (n-6).In some embodiments, the compositions may comprise at least about 10 wt.% ethyl ester of docosahexaenoic acid and at least about 10 wt. % ethylester of docosapentaenoic acid (n-6). In other embodiments, thecompositions may comprise at least about 15 or 20 wt. % ethyl ester ofdocosahexaenoic acid and at least about 15 or 20 wt. % ethyl ester ofdocosapentaenoic acid (n-6).

The present invention also provides compositions comprising at leastabout 90 wt. % of a combination of ethyl ester of docosahexaenoic acidand ethyl ester of docosapentaenoic acid (n-6), and at least oneadditional fatty acid or an ester thereof. In certain embodiments, thecompositions may comprise at least about 91, 92, 93, 94, 95, 96, 97, 98,or 99 wt. % of a combination of ethyl ester of docosahexaenoic acid andethyl ester of docosapentaenoic acid (n-6). In some embodiments, theadditional fatty acid may have a boiling point of about 150-170° C. at apressure of 0.8 mm Hg.

The DHA/DPA (n-6) compositions described above may further comprise lessthan about 4% of a saturated fatty acid or an ester thereof. In certainembodiments, the compositions may comprise less than about 3.5%, 3.0%,2.5%, 2.0%, 1.5%, 1.0% or 0.5% of a saturated fatty acid or an esterthereof.

In some embodiments, the saturated fatty acid or an ester thereof maycontain less than 20 carbons, such as, for example, a saturated fattyacid or an ester thereof that contains 19, 18, 17. 16, 15, 14, 13, 12,11, 10, 9 or 8 carbons. In certain embodiments, the saturated fatty acidor ester thereof may contain 14 or 16 carbons.

PUFA esters and compositions of the present invention (at times referredto collectively as “PUFA esters”) may be used in pharmaceuticalproducts. In some embodiments, the pharmaceutical products may containPUFA esters without an additional pharmaceutically active agent. Inother embodiments, the pharmaceutical product may comprise apharmaceutically active agent. Examples of pharmaceutically activeagents include statins, anti-hypertensive agents, anti-diabetic agents,anti-dementia agents, anti-depressants, anti-obesity agents, appetitesuppressants and agents to enhance memory and/or cognitive function. Thepharmaceutical products may further comprises any pharmaceuticallyacceptable excipient, carriers, binders or other formulation componentsknown in the art.

PUFA esters produced by the methods of the present invention andcompositions of the present invention are suitable for use astherapeutic and experimental agents. An embodiment of the presentinvention comprises the production of PUFA esters for treatment ofPUFA-deficient infants. The PUFA esters can be included in a parenteralformulation that can be administered to an infant through parenteralroutes to fortify the infant's supply of a PUFA. Preferred parenteralroutes include, but are not limited to, subcutaneous, intradermal,intravenous, intramuscular and intraperitoneal routes. A parenteralformulation can include PUFA esters of the present invention and acarrier suitable for parenteral delivery. As used herein, a “carrier”refers to any substance suitable as a vehicle for delivering a moleculeor composition to a suitable in vivo site of action. Examples of suchcarriers include, but are not limited to water, phosphate bufferedsaline, Ringer's solution, dextrose solution, serum-containingsolutions, Hank's solution and other aqueous physiologically balancedsolutions. Suitable carriers also include oil-based carriers,non-aqueous solutions, suspensions, and emulsions. Examples includepropylene glycol, polyethylene glycol, vegetable oils such as olive oil,injectable organic esters such as ethyl oleate, polyethoxylated castoroil (cremaphor), and others known in the art. Acceptable protocols toadminister PUFA esters in an effective manner include individual dosesize, number of doses, frequency of dose administration, and mode ofadministration. Determination of such protocols can be accomplished bythose skilled in the art depending upon a variety of variables,including the weight of the infant and the extent of PUFA deficiency.Another embodiment of the present invention comprises the production ofPUFA esters for treatment of adults, in particular pregnant mothers. Theproduct may be used for augmenting long chain PUFA levels in milk ofwarm-blooded animals. Acceptable protocols for administration of PUFAesters to adults includes parenteral feeding techniques or encapsulatingPUFA esters of the present invention in a capsule, such as gelatin(i.e., digestible) capsule, for oral administration and/or in a liquiddiet formulation. A liquid diet formulation can comprise a liquidcomposition containing nutrients suitable for supplementing a diet ornutrients sufficient as a complete diet.

PUFA esters produced by the methods of the present invention andcompositions of the present invention may also be used to treat subjects(e.g., humans or animals) with high levels of triglycerides, includingsubjects with triglyceridemia. For example, subjects with fastingtriglycerides of 150 mg/dL or above may benefit from treatment with thePUFA esters of the present invention, and, additionally, the elevationof post-parandial triglyercides may be reduced by treatment with thePUFA esters of the present invention. In some embodiments, individualPUFA esters may be administered to a subject to treat high levels oftriglycerides. In certain embodiments, the PUFA ester may be DHA or ARA.In other embodiments combinations of PUFA esters may be administered toa subject to treat high levels of triglycerides. In certain embodiments,the combination of PUFA esters may comprise omega-3 and omega-6 PUFASsuch as DHA and DPA n-6. In some embodiments, the PUFA esters maycomprise about 90% of a composition administered to the subject. ThePUFA esters may be administered with other components and excipients,such as the carriers described above. The PUFA esters may also be usedto treat subjects with diseases that can be associated with high levelsof triglycerides, such as cardiovascular disease or hypertension.

PUFA esters and compositions of the present invention may be used totreat subjects with neurological disorders, dementia and pre-dementiarelated conditions. These conditions include Alzheimer's Disease,Vascular Dementia, Mixed Dementia, Dementia with Lewy Bodies, as well assecondary dementias caused by drugs, delirium, or depression.

Therapeutic compounds appropriate to use with the PUFA esters andcompositions of the present invention include any therapeutic which canbe used to protect an individual against any of the conditions ordiseases discussed herein, and may include a protein, an amino acid, adrug, other natural products and a carbohydrate. Such therapeuticcompounds will be well known to those of skill in the art for theparticular disease or condition being treated. Some preferredtherapeutic compounds to combine with a composition or formulation ofthe invention include, but are not limited to: Tacrine (COGNEX);Donepezil (ARICEPT); Rivastigmine (EXELON); Galantamine (REMINYL);Memantine (AKATINOL); Neotropin; Nootropics; Alpha-tocopherol (vitaminE); Selegeline (ELDEPRYL); non-steroidal anti-inflammatory agents(NSAIDS); Gingko biloba; estrogen; β-secretase inhibitors; vaccines,including lipid or liposome-based vaccines, that dissolve plaques in thebrain; B complex vitamins; calcium channel blockers; HMG CoA reductaseinhibitors; statins and other anti-cholesterol drugs (e.g., ZOCOR(simvastatin), LIPITOR (atorvastatin calcium), LESCOL (fluvastatin),LOPID (gemfibrozil), or PRAVACHOL (pravastatin sodium)); policosanols;fibrates; Clioquinol; (and other natural products (e.g., curcumin,lignans, phytoestrogens, phytosterols; niacin, and vitamin supplements).

Dosages and routes of administration are known in the art and may bedetermined by those of skill in the art.

Although PUFA esters and compositions of the present invention can beadministered topically or as an injectable, the most preferred route ofadministration is oral administration. The PUFAs may be administered toindividuals in the form of nutritional supplements and/or foods and/orpharmaceutical formulations and/or beverages. A preferred type of foodis a medical food (e.g., a food which is in a formulation to be consumedor administered externally under the supervision of a physician andwhich is intended for the specific dietary management of a disease orcondition for which distinctive nutritional requirements, based onrecognized scientific principles, are established by medicalevaluation.) For infants, the fatty acids are administered to infants asinfant formula, weaning foods, jarred baby foods, human milk fortifierand/or infant cereals.

Any biologically acceptable dosage forms, and combinations thereof, arecontemplated by the inventive subject matter. Examples of such dosageforms include, without limitation, chewable tablets, quick dissolvetablets, effervescent tablets, reconstitutable powders, elixirs,liquids, solutions, suspensions, emulsions, tablets, multi-layertablets, bi-layer tablets, capsules, soft gelatin capsules, hard gelatincapsules, caplets, lozenges, chewable lozenges, beads, powders,granules, particles, microparticles, dispersible granules, cachets,douches, suppositories, creams, topicals, inhalants, aerosol inhalants,patches, particle inhalants, implants, depot implants, ingestibles,injectables, infusions, health bars, confections, cereals, cerealcoatings, foods, nutritive foods, functional foods and combinationsthereof. The preparations of the above dosage forms are well known topersons of ordinary skill in the art. Preferably, a food that isenriched with the desired PUFA is selected from the group including, butnot limited to: baked goods and mixes; chewing gum; breakfast cereals;cheese products; nuts and nut-based products; gelatins, pudding, andfillings; frozen dairy products; milk products; dairy product analogs;soft candy; soups and soup mixes; snack foods; processed fruit juice;processed vegetable juice; fats and oils; fish products; plant proteinproducts; poultry products; and meat products.

The present invention also includes a method of making any of theabove-described compositions of the invention, such as by combining thecomponents of the composition into any suitable delivery form using anysuitable method known in the art.

According to the present invention, the methods of the present inventionare suitable for use in an individual that is a member of the Vertebrateclass, Mammalia, including, without limitation, primates, livestock anddomestic pets (e.g., a companion animal). Most typically, an individualwill be a human individual. The term “individual” can be interchangedwith the term “subject” or “patient” and refers to the subject of aprotocol or method according to the invention. Accordingly, anindividual can include a healthy, normal (non-diseased) individual, aswell as an individual who has or is at risk of developing pre-dementiaor dementia or a symptom or indicator thereof as described herein.

The PUFA esters produced by the methods of the present invention may beused to produce PUFA salts. In some embodiments, PUFA salts can beproduced by reacting the PUFA esters of the present invention in thepresence of an alkaline metal base such as an alkaline metal hydroxide(e.g., potassium hydroxide). The PUFA salts formed from the PUFA estersof the present invention can be used in a variety of applications, suchas in foods, beverages, and pharmaceuticals. In some embodiments, thePUFA salts produced using the PUFA esters of the present invention arewater-soluble and can be used directly in foods, beverages, andpharmaceuticals.

PUFA esters produced by the methods of the present invention can be usedin any animal food material, particularly food materials for humans, tocreate a food product having enhanced concentrations of PUFAs. Theamount of fatty acids naturally in food products varies from one foodproduct to another. A food product of the present invention can have anormal amount of a PUFA or a modified amount of a PUFA. In the formerinstance, a portion of the naturally occurring lipids may be substitutedby PUFA esters of the present invention. In the latter instance,naturally occurring lipids may be supplemented by PUFA esters of thepresent invention.

PUFA esters may be added to foods for infants, such as infant formulaand baby food. According to the present invention, an infant refers toinfants and children less than about two years old, including, inparticular, premature infants. Certain PUFAs are particularly importantcomponent of infant formula and baby food because of the rapid growth ofinfants (i.e., doubling or tripling in weight during the first year oflife). An effective amount of PUFA ester to supplement infant formula isan amount that approximates the concentration of the PUFAs in humanbreast milk. Preferred amounts of PUFA esters to add to infant formulaor baby food range from between about 0.1 to about 1.0% of total fattyacids, more preferably from between about 0.1 to about 0.6% of totalfatty acids, and even more preferably about 0.4% of total fatty acids.

Another aspect of the present invention includes a food productcomprising a food material combined with PUFA esters of the presentinvention. PUFA esters may be added to a food material to create a foodproduct having enhanced concentrations of PUFAs. As used herein, theterm “food material” refers to any food type fed to humans or non-humananimals. Also within the scope of the present invention is a method tomake a food product comprising adding PUFA esters produced by methods ofthe present invention to a food material.

A suitable food material useful for the formation of a food product ofthe present invention includes animal food. The term “animal” means anyorganism belonging to the kingdom Animalia and includes, withoutlimitation, primates (e.g., humans and monkeys), livestock and domesticpets. The term “food product” includes any product to be fed to suchanimals. Preferred food materials to be consumed by humans includeinfant formula and baby food. Preferred food materials to be consumed bydomestic pets include dog foods.

PUFA esters produced by methods of the present invention can be added toa wide range of products such as baked goods, vitamin supplements, dietsupplements, powdered drinks, etc. at various stages of production.Numerous finished or semi-finished powdered food products can beproduced using the compositions of the present invention.

A partial list of food products comprising the products of the presentinvention includes doughs, batters, baked food items including, forexample, such items as cakes, cheesecakes, pies, cupcakes, cookies,bars, breads, rolls, biscuits, muffins, pastries, scones, and croutons;liquid food products, for example, beverages, energy drinks, infantformula, liquid meals, fruit juices, multivitamin syrups, mealreplacers, medicinal foods, and syrups; semi-solid food products such asbaby food, yoghurt, cheese, cereal, pancake mixes; food bars includingenergy bars; processed meats; ice creams; frozen desserts; frozenyoghurts; waffle mixes; salad dressings; and replacement egg mixes. Alsoincluded are baked goods such as cookies, crackers, sweet goods, snackcakes, pies, granola/snack bars, and toaster pastries; salted snackssuch as potato chips, corn chips, tortilla chips, extruded snacks,popcorn, pretzels, potato crisps, and nuts; specialty snacks such asdips, dried fruit snacks, meat snacks, pork rinds, health food bars andrice/corn cakes; and confectionary snacks such as candy.

The present invention, while disclosed in terms of specific methods,products, and organisms, is intended to include all such methods,products, and organisms obtainable and useful according to the teachingsdisclosed herein, including all such substitutions, modifications, andoptimizations as would be available to those of ordinary skill in theart. The following examples and test results are provided for thepurposes of illustration and are not intended to limit the scope of theinvention.

EXAMPLES Example 1

This example illustrates a method of the present invention for purifyingethyl docosahexaneoate (DHA ethyl ester) from docosahexaneoicacid-containing single cell oil.

150 mL of absolute ethanol (EtOH) was added to 175 g (approximately 0.2moles of triglyceride) of DHASCO®-T oil (Martek Biosciences Corporation,Columbia, Md., having a DHA content of 0.4 g/g oil) in a one-liter flaskunder nitrogen (N₂) at room temperature. DHASCO®-T oil is prepared fromthe microalgae Crypthecodinium cohnii. The mixture was allowed to stirfor 15 minutes to obtain a homogeneous solution. 67 g of a 21% solutionof sodium ethoxide/ethanol (NaOEt/EtOH; approximately 1.04 molarequivalents of triglycerides) was then added to the solution and themixture was allowed to reflux under N₂ for about 9 hours. The progressof the reaction was monitored by gas chromatography (GC) and thin-layerchromatography (TLC). When the reaction was completed, approximately 75mL of EtOH was removed by distillation. The reaction mixture was thenallowed to cool to room temperature under N₂. 300 mL hexane was added tothe cooled reaction mixture, and the mixture was allowed to stir for 15minutes at room temperature. 300 mL of deionized water was then added tothe mixture, and the mixture was allowed to stir for an additional 15minutes. After removing and saving the organic layer, the aqueous layerwas washed twice with 300 mL portions of hexane. A dark brown aqueouslayer was discarded. The combined organic layers were then washed with200 mL of a saturated NaCl solution. A GC analysis of the organic layerindicated the presence of about 44.7% DHA ethyl ester; the remainingmaterials were predominantly lower molecular weight ethyl esters (seeTable 1).

The combined organic layer was concentrated under reduced pressure. Thecrude concentrate was then subjected to vacuum fractional distillation.The lower molecular weight ethyl esters were collected at temperaturesbetween 100-150° C. and at a pressure of 0.8 mm Hg. The major componentsof this fraction were oleic, saturated C-14, and C-12 esters. The DHAethyl ester was collected at temperatures between 155-165° C. and at apressure of 0.8 mm Hg. A GC analysis of the DHA ethyl ester fractionshowed a purity of about 91.3% DHA (see Table 1). From the fractionaldistillation, 68 g (86% yield) of the DHA ethyl ester was obtained as alight yellow oil.

TABLE 1 GC Analyses of DHASCO ®-T Oil Transesterification andDistillation Products DHA Ethyl Ester-Containing Organic Layer AfterFraction After Vacuum Sample Transesterifaction Fractional Distillation% 22:6 (n-3) DHA 44.72 91.29 % 20:5 (n-3) EPA 0.00 0.00 % Additional55.28 8.81 components

Example 2

This example illustrates a method of the present invention for purifyingethyl docosahexaneoate (DHA ethyl ester) from a crude Crypthecodiniumcohnii oil.

A crude oil obtained from Crypthecodinium cohnii by hexane extraction(DHA content of 0.5 g/g oil) was used directly without any furtherprocessing, such as winterization and/or RBD processing. 150 mL ofabsolute ethanol was added to 175 g (approximately 0.2 moles oftriglycerides) of the crude oil in a one-liter flask under N₂ at roomtemperature. The mixture was allowed to stir for 15 minutes to obtain ahomogeneous solution. 67 g of a 21% solution of NaOEt/EtOH(approximately 1.04 molar equivalents of triglycerides) was then addedto the solution, and the mixture was allowed to reflux under N₂ forabout 10 hours. The progress of the reaction was monitored by GC andTLC. When the reaction was completed, approximately 75 mL of ethanol wasremoved by distillation, and the mixture was allowed to cool to roomtemperature under N₂. 300 mL of hexane was added to the cooled mixture,and the mixture was allowed to stir for 15 minutes at room temperature.300 mL of deionized water was then added to the mixture, and the mixturewas allowed to stir for an additional 15 minutes. After removing andsaving the organic layer, the aqueous layer was washed twice with 300 mLportions of hexane. The combined organic layer was then washed with 200mL of a saturated NaCl solution. A GC analysis of the organic layerindicated the presence of about 51% DHA ethyl ester; the remainingmaterials were predominantly lower molecular weight ethyl esters (seeTable 2).

The combined organic layer was concentrated under reduced pressure. Thecrude concentrate was then subjected to vacuum fractional distillation.The lower molecular weight ethyl esters were collected at temperaturesbetween 100-150° C. and at a pressure of 0.8 mm Hg. The major componentsof this fraction were oleic, saturated C-14, and C-12 esters. The DHAethyl ester was collected at temperatures between 155-165° C. and at apressure of 0.8 mm Hg. A GC analysis of the DHA ethyl ester fractionshowed a purity of about 92% DHA (see Table 2). From the fractionaldistillation, 69 g (66% yield) of the DHA ethyl ester was obtained as alight yellow oil.

TABLE 2 GC Analyses of Crude Crypthecodinium cohnii OilTransesterification and Distillation Products DHA Ethyl Ester-ContainingOrganic Layer After Fraction After Vacuum Sample TransesterifactionFractional Distillation % 22:6 (n-3) DHA 51.25 91.80 % 20:5 (n-3) EPA0.00 0.00 % Additional 48.75 8.20 components

Example 3

This example illustrates a method of the present invention for purifyingethyl docosahexaenoate (as a DHA ethyl ester/DPA ethyl ester mixture)from a crude Schizochytrium sp. oil.

A crude oil obtained from Schizochytrium sp. by hexane extraction wasused directly without any further processing, such as winterizationand/or RBD processing.

150 mL of absolute ethanol was added to 175 g (approximately 0.2 molesof triglycerides) of the crude oil (DHA content 40%, DPA content 15%) ina one-liter flask under N₂ at room temperature. The mixture was allowedto stir for 15 minutes to obtain a homogeneous solution. 67 g of a 21%solution of NaOEt/EtOH (approximately 1.04 molar equivalents oftriglycerides) was then added to the solution, and the mixture wasallowed to reflux under N₂ for about 10 hours. The progress of thereaction was monitored by GC and TLC. When the reaction was completed,approximately 65 mL of ethanol was removed by distillation, and themixture was allowed to cool to room temperature under N₂. 300 mL ofhexane was added to the cooled mixture, and the mixture was allowed tostir for 15 minutes at room temperature. 300 mL of deionized water wasthen added to the mixture, and the mixture was allowed to stir for anadditional 15 minutes. After removing and saving the organic layer, theaqueous layer was washed twice with 300 mL portions of hexane. Thecombined organic layer was washed with 200 mL of a saturated NaClsolution. A GC analysis of the organic indicated the presence of about40% DHA ethyl ester and 15% DPA ethyl ester; the remaining materialswere predominantly lower molecular weight ethyl esters (see Table 3).

The combined organic layer was concentrated under reduced pressure. Thecrude concentrate was then subjected to vacuum fractional distillation.The lower molecular weight ethyl esters were collected at temperaturesbetween 100-150° C. and at a pressure of 0.8 mm Hg. The major componentsof this fraction were saturated C-14, and C-16 ethyl esters. The DHAethyl ester/DPA ethyl ester mixture was collected at temperaturesbetween 155-170° C. and at a pressure of about 0.5 mm Hg. A GC analysisof the DHA/DPA ethyl ester fraction showed a combined purity of about93% (see Table 3). From the fractional distillation, 85 g (85% yield) ofthe DHA/DPA ethyl ester was obtained as a very light yellow oil.

TABLE 3 GC Analyses of Crude Schizochytrium sp. Oil Transesterificationand Distillation Products DHA/DPA Ethyl Ester- Containing FractionOrganic Layer After After Vacuum Fractional Sample TransesterifactionDistillation % 22:6 (n-3) DHA 40.07 67.31 % 22:5 (n-6) DPA 15.09 25.86 %20:5 (n-3) EPA 1.21 0.32 % Additional 43.63 6.5 components

Example 4

This example illustrates GC analyses of crude and purified PUFA ethylesters from Crypthecodinium cohnii oil and Schizochytrium sp. oil.

A crude oil obtained from Schizochytrium sp. or Crypthecodinium cohniiby hexane extraction was used directly without any further processing,such as winterization and/or RBD processing. The crude oils were thensubjected to a transesterification reaction as described above inExamples 2 and 3. The crude ethyl esters were then subjected to ureaadduction as described above, or to distillation as described inExamples 2 and 3. GC analyses were then performed on each sample alongwith a DPA ethyl ester product or DHA ethyl ester product (Nu-Chek Prep,Inc., Elysian, Minn.). The results are presented below in Table 4(Schizochytrium sp.) or Table 5 (Crypthecodinium cohnii). Two analyseswere performed on the crude and distilled ethyl esters from theCrypthecodinium cohnii oil.

TABLE 4 GC Analyses of Schizochytrium sp. Ethyl Ester Products EthylEsters Ethyl Esters after Urea after Crude Adduction Distillation Ethylof Crude of Crude Nu-Chek Fatty Acid Esters Oil Oil DPA EE % C12:0 0.260.20 0.00 0.67 % C14:0 8.63 1.68 0.00 0.00 % C14:1 0.00 0.13 0.00 0.36 %C16:0 24.65 0.53 0.00 0.10 % C16:1 0.40 0.00 0.00 0.00 % C18:0 0.57 0.000.00 0.10 % C18:1 (n-9) 0.36 0.00 0.00 1.77 % C18:1 (n-7) 0.35 0.00 0.000.72 % C18:2 0.41 0.00 0.00 0.00 % C18:2 (n-6) 0.24 0.58 0.00 0.00 %C20:3 (n-6) 0.42 2.75 0.00 0.00 % C20:3 (n-3) 0.00 0.27 0.00 0.00 %C20:3 (n-6) 0.00 0.59 0.00 0.00 % C20:4 ARA 1.50 2.29 0.00 0.16 % C20:5(n-3) EPA 0.00 2.01 0.00 0.00 % C22:4 (n-6) 0.00 0.00 0.00 3.83 % C22:5(n-6) DPA 15.89 22.78 26.46 87.15 % C22:5 (n-3) DPA 5.21 % C22:6 (n-3)DHA 40.65 57.67 71.83 0.00 % Additional 4.95 7.19 0.94 0.80 components

Example 5

This example illustrates a method of the present invention for purifyingethyl docosahexaneoate (DHA ethyl ester) from a mixture of fatty acidethyl esters of docosahexaneoic acid-containing single cell oil via ureacrystallization.

150 g crude mixture of fatty acid ethyl esters, obtained bytransesterification of docosahexaneoic acid-containing single cellDHASCO®-T oil prepared from the microalgae Crypthecodinium cohnii, wasadded to a solution of 262.5 g urea (1.75 wt. eq. of esters) in 1050 mLmethanol (7 vol eq of esters) at 70° C. under nitrogen. The resultingmixture of urea and esters was continued to heat at 70° C. undernitrogen for 1 hr. The mixture was first allowed to cool to 20° C.followed by cooling to 0-4° C. to complete the urea adductcrystallization. The mixture was allowed to stand for additional 2 hoursat 0-4° C. The crystallized urea adduct was then filtered at 0-4° C.

The filtrate was diluted with 300 mL of water and the mixture wasacidified with dilute sulfuric acid to a pH of 1-2. The acidifiedsolution was extracted with 300 mL×3 of hexane. The combined hexaneextracts were washed with saturated NaCl solution. The washed hexanesolution was dried over anhydrous sodium sulfate and concentrated invacuo to obtain 70-75% of theoretical yield. Typically, GC analysisshowed purity of above obtained DHA Ethyl ester around 90-96% (800-860mg/g).

TABLE 5 GC Analyses of Crypthecodinium cohnii Ethyl Ester Products EthylEsters Ethyl Ethyl after Urea Esters after Crude Esters after CrudeAdduction Distillation Ethyl Distillation Ethyl of Crude of Crude Estersof Crude Oil Nu-Chek Fatty Acid Esters Oil Oil #2 #2 DHA EE % C8:0 0.190.00 0.00 0.36 0.00 0.00 % C10:0 0.95 0.00 0.00 1.86 0.00 0.00 % C12:04.04 0.00 0.00 7.18 0.00 0.00 % C13:0 0.00 0.09 0.00 0.00 0.00 0.00 %C14:0 13.77 0.00 0.00 19.99 0.00 0.00 % C14:1 0.17 0.00 0.00 0.19 0.000.00 % C16:0 11.08 0.00 0.10 16.15 0.00 0.10 % C16:1 2.83 0.00 0.00 2.240.00 0.00 % C18:0 0.21 0.00 0.00 0.48 0.00 0.00 % C18:1 (n-9) 9.77 0.000.00 10.27 0.00 0.00 % C18:1 (n-7) 0.00 0.00 0.00 0.00 0.18 0.00 % C22:5(n-3) 0.65 0.00 1.16 0.20 0.59 0.00 DPA % C22:6 (n-3) 55.64 93.00 89.6741.26 96.73 99.95 DHA % C24:0 0.00 0.00 0.69 0.00 0.00 0.00 % C28:8 0.60** 1.40 0.25 1.20 0.00 % Additional 0.70 2.52 8.38 0.50 1.11 0.05components ** Not Determined

The principles, preferred embodiments and modes of operation of thepresent invention have been described in the foregoing specification.The invention which is intended to be protected herein should not,however, be construed as limited to the particular forms disclosed, asthese are to be regarded as illustrative rather than restrictive.Variations and changes may be made by those skilled in the art withoutdeparting from the spirit of the present invention. Accordingly, theforegoing best mode of carrying out the invention should be consideredexemplary in nature and not as limiting to the scope and spirit of theinvention as set forth in the appended claims.

1. A method for purifying a composition comprising triglycerides havingpolyunsaturated fatty acid residues comprising: a) reacting thecomposition in the presence of an alcohol and a base to produce an esterof a polyunsaturated fatty acid from the triglycerides; and b)distilling the composition to recover a fraction comprising the ester ofthe polyunsaturated fatty acid.
 2. The method of claim 1, wherein thestep of reacting the composition in the presence of an alcohol and abase is performed at a temperature from about 60° C. to about 120° C. 3.The method of claim 1, wherein the step of reacting the composition inthe presence of an alcohol and a base is performed for a time from about2 hours to about 12 hours.
 4. The method of claim 1, wherein thecomposition comprising triglycerides having polyunsaturated fatty acidresidues has not been subjected to one or more treatments selected fromthe group consisting of refining, desolventization, deodorization,winterization, chill filtration, and bleaching.
 5. The method of claim1, wherein the composition comprising triglycerides havingpolyunsaturated fatty acid residues has not been subjected to refining,desolventization, deodorization, winterization, chill filtration, andbleaching.
 6. The method of claim 1, wherein the composition comprisingtriglycerides having polyunsaturated fatty acid residues is from asource selected from the group consisting of a plant, a microorganism,an animal, and mixtures of the foregoing.
 7. The method of claim 6,wherein the source is a microorganism selected from the group consistingof algae, bacteria, fungi and protists.
 8. The method of claim 6,wherein the source is selected from the group consisting of plantsselected from the group consisting of soybean, corn, rice, safflower,sunflower, canola, flax, peanut, mustard, rapeseed, chickpea, cotton,lentil, white clover, olive, palm, borage, evening primrose, linseed andtobacco and mixtures thereof.
 9. The method of claim 6, wherein thesource is selected from the group consisting of a genetically modifiedplant and a genetically modified microorganism, wherein the geneticmodification comprises the introduction of polyketide synthase genes.10. The method of claim 6, wherein the source is a microorganismselected from the group consisting of Thraustochytriales,dinoflagellates, and Mortierella.
 11. The method of claim 10, whereinthe microorganism is Thraustochytriales.
 12. The method of claim 11,wherein the microorganism is Schizochytrium.
 13. The method of claim 11,wherein the microorganism is Thraustochytrium.
 14. The method of claim10, wherein the microorganism is a dinoflagellate of the genusCrypthecodinium.
 15. The method of claim 6, wherein the source is ananimal selected from aquatic animals.
 16. The method of claim 1, whereinthe polyunsaturated fatty acid is a polyunsaturated fatty acid having achain length of at least 18 carbons.
 17. The method of claim 1, whereinthe polyunsaturated fatty acid is a polyunsaturated fatty acid selectedfrom the group consisting of docosahexaenoic acid, docosapentaenoicacid, arachidonic acid, eicosapentaenoic acid, stearidonic acid,linolenic acid, alpha linolenic acid, gamma linolenic acid, conjugatedlinolenic acid and mixtures thereof.
 18. The method of claim 17, whereinthe polyunsaturated fatty acid is docosahexaenoic acid.
 19. The methodof claim 17, wherein the polyunsaturated fatty acid is arachadonic acid.20. The method of claim 1, wherein the base is a base of the formulaRO-M, wherein M is a monovalent cation and RO is an alkoxide of a C₁₋₆alkyl alcohol.
 21. The method of claim 1, wherein the base is sodiumethoxide.
 22. The method of claim 1, wherein the alcohol is a C₁₋₆ alkylalcohol.
 23. The method of claim 1, wherein the alcohol is ethanol andthe ester is an ethyl ester of the polyunsaturated fatty acid.
 24. Themethod of claim 1, wherein the step of distilling the composition torecover a fraction comprising the ester of the polyunsaturated fattyacid is performed under vacuum.
 25. The method of claim 24, wherein thestep of distilling the composition to recover a fraction comprising theester of the polyunsaturated fatty acid is performed at a temperature ofless than about 170° C.
 26. The method of claim 1, wherein the fractionrecovered comprises at least about 50 wt. % ester of the polyunsaturatedfatty acid.
 27. The method of claim 1, wherein the fraction recoveredcomprises at least about 75 wt. % ester of the polyunsaturated fattyacid.
 28. The method of claim 1, wherein the fraction recoveredcomprises at least about 90 wt. % ester of the polyunsaturated fattyacid.
 29. The method of claim 1, wherein the fraction recoveredcomprises at least about 95 wt. % ester of the polyunsaturated fattyacid.
 30. The method of claim 1, wherein the step of reacting thecomposition in the presence of an alcohol and a base produces an esterof a polyunsaturated fatty acid from the triglycerides by directtransesterification.
 31. The method of claim 1, further comprising: a)combining the fraction comprising the ester of the polyunsaturated fattyacid with urea in a medium; b) cooling or concentrating the medium toform a urea-containing precipitate and a liquid fraction; and c)separating the precipitate from the liquid fraction.
 32. The method ofclaim 31, wherein the medium further comprises an organic solvent thatcan solubilize the ester of the polyunsaturated fatty acid.
 33. Themethod of claim 32, wherein the organic solvent comprises an alkylalcohol comprising from 1 to 4 carbon atoms.
 34. The method of claim 33,wherein the organic solvent comprises ethanol.
 35. The method of claim31, wherein the medium is cooled to a temperature of from about 0° C. toabout 25° C. to form the urea-containing precipitate.
 36. The method ofclaim 31, wherein at least a portion of the urea-containing precipitateis formed under a non-oxidizing atmosphere.
 37. A method for producingan ester of a polyunsaturated fatty acid from a composition comprisingtriglycerides having polyunsaturated fatty acid residues comprising: a)transesterifying the composition in the presence of an alcohol and abase to produce an ester of the polyunsaturated fatty acid from thetriglycerides; and b) distilling the composition to recover a fractioncomprising the ester of the polyunsaturated fatty acid.
 38. (canceled)39. A method for preparing a composition comprising an ester of apolyunsaturated fatty acid comprising: reacting a composition comprisingtriglycerides having polyunsaturated fatty acid residues in the presenceof an alcohol and a base to produce an ester of a polyunsaturated fattyacid from the triglycerides, wherein the composition comprisingtriglycerides having polyunsaturated fatty acid residues has not beensubjected to one or more treatments selected from the group consistingof refining, desolventization, deodorization, winterization, chillfiltration, and bleaching. 40-67. (canceled)
 68. A compositioncomprising at least about 90 wt. % ethyl ester of docosahexaenoic acid,wherein the composition further comprises at least about 0.1 wt. % of4,7,10,13,16,19,22,25 octacosaoctaenoic acid (C28:8) or an esterthereof.
 69. The composition of claim 68, wherein the compositioncomprises at least about 0.5 wt. % of 4,7,10,13,16,19,22,25octacosaoctaenoic acid (C28:8) or an ester thereof.
 70. The compositionof claim 68, wherein the composition comprises at least about 1.0 wt. %of 4,7,10,13,16,19,22,25 octacosaoctaenoic acid (C28:8) or an esterthereof.
 71. The composition of claim 68, wherein the compositioncomprises at least about 1.2 wt. % of 4,7,10,13,16,19,22,25octacosaoctaenoic acid (C28:8) or an ester thereof.
 72. The compositionof claim 68, wherein the composition further comprises at least about0.1 wt. % of docosapentaenoic acid (n-3) or an ester thereof.
 73. Thecomposition of claim 68, wherein the composition further comprises atleast about 0.3 wt. % of docosapentaenoic acid (n-3) or an esterthereof.
 74. The composition of claim 68, wherein the compositionfurther comprises at least about 0.4 wt. % of docosapentaenoic acid(n-3) or an ester thereof.
 75. The composition of claim 68, wherein thecomposition further comprises at least about 0.5 wt. % ofdocosapentaenoic acid (n-3) or an ester thereof.
 76. The composition ofclaim 68, wherein the composition comprises at least about 92 wt. %ethyl ester of docosahexaenoic acid.
 77. The composition of claim 68,wherein the composition comprises at least about 95 wt. % ethyl ester ofdocosahexaenoic acid.
 78. The composition of claim 68, wherein thecomposition further comprises less than about 1 wt. % eicosapentaenoicacid or an ester thereof.
 79. The composition of claim 68, wherein thecomposition further comprises less than about 0.5 wt. % eicosapentaenoicacid or an ester thereof.
 80. The composition of claim 68, wherein thecomposition further comprises less than about 0.25 wt. %eicosapentaenoic acid or an ester thereof.
 81. A composition comprisingat least about 90 wt. % ethyl ester of docosahexaenoic acid, wherein thecomposition further comprises at least about 0.1 wt. % ofdocosapentaenoic acid (n-3) or an ester thereof.
 82. The composition ofclaim 81, wherein the composition comprises at least about 0.3 wt. % ofdocosapentaenoic acid (n-3) or an ester thereof.
 83. The composition ofclaim 81, wherein the composition comprises at least about 0.4 wt. % ofdocosapentaenoic acid (n-3) or an ester thereof.
 84. The composition ofclaim 81, wherein the composition comprises at least about 0.5 wt. % ofdocosapentaenoic acid (n-3) or an ester thereof.
 85. The composition ofclaim 81, wherein the composition further comprises at least about 0.5wt. % of 4,7,10,13,16,19,22,25 octacosaoctaenoic acid (C28:8) or anester thereof.
 86. The composition of claim 81, wherein the compositionfurther comprises at least about 0.75 wt. % of 4,7,10,13,16,19,22,25octacosaoctaenoic acid (C28:8) or an ester thereof.
 87. The compositionof claim 81, wherein the composition further comprises at least about1.0 wt. % of 4,7,10,13,16,19,22,25 octacosaoctaenoic acid (C28:8) or anester thereof.
 88. The composition of claim 81, wherein the compositionfurther comprises at least about 1.2 wt. % of 4,7,10,13,16,19,22,25octacosaoctaenoic acid (C28:8) or an ester thereof.
 89. The compositionof claim 81, wherein the composition comprises at least about 92 wt. %ethyl ester of docosahexaenoic acid.
 90. The composition of claim 81,wherein the composition comprises at least about 95 wt. % ethyl ester ofdocosahexaenoic acid.
 91. The composition of claim 81, wherein thecomposition further comprises less than about 1 wt. % eicosapentaenoicacid or an ester thereof.
 92. The composition of claim 81, wherein thecomposition further comprises less than about 0.5 wt. % eicosapentaenoicacid or an ester thereof.
 93. The composition of claim 81, wherein thecomposition further comprises less than about 0.25 wt. %eicosapentaenoic acid or an ester thereof.
 94. A composition comprisingat least about 90 wt. % ethyl ester of docosahexaenoic acid, wherein thecomposition further comprises at least one additional fatty acid or anester thereof with a boiling point of about 150-170° C. at a pressure of0.8 mm Hg. 95-98. (canceled)
 99. A composition comprising at least about90 wt. % of a combination of ethyl ester of docosahexaenoic acid andethyl ester of docosapentaenoic acid (n-6). 100-109. (canceled)
 110. Amethod of treating a subject with high levels of triglyceridescomprising administering a composition according to claim 68, 81, 94, or99 to the subject.
 111. A method of treating a subject with aneurological disorder, dementia or a pre-dementia related conditioncomprising administering a composition according to claim 68, 81, 94, or99 to the subject.